Time-dependent shift from antagonism to synergism in mixture toxicity mediated by GFP-modified Caenorhabditis elegans

Environmental contaminant mixtures present complex toxicological challenges, especially in transgenic organisms where genetic modifications may alter toxicity mechanisms. This study addresses the uncharacterized influence of GFP fusion proteins on mixture interactions using Caenorhabditis elegans. We employed uniform design ray methodology to evaluate 6 compounds, including ionic liquids (ILs) such as 1-butylpyridinium chloride ([bpy]Cl) and 1-butylpyridinium bromide ([bpy]Br), pesticides (dichlorvos and glyphosate), and substituted phenols (4-chlorophenol and 4-nitrophenol), in transgenic DAF-16::GFP (DAF-16) versus wild-type N2 strains. Toxicity interactions were quantified using the improved combination index (CI_imp) with 95% observation confidence intervals (OCIs) at 12 h and 24 h. Results showed that while ILs exhibited negligible lethality in N2, they induced marked toxicity in DAF-16. The transgenic strain demonstrated 10-100 times greater lethality to the mixtures compared to N2, with 95% OCIs confirming significant leftward shifts in concentration-response curves. A time-dependent interaction shift occurred: initial antagonism at 12 h (CI_imp 1.315-7.340) transitioned to significant synergism exclusively in the DAF-16 strain at 24 h (CI_imp <1; 95% OCIs excluded 1). Molecular docking provided a preliminary structural hypothesis, suggesting that GFP fusion might alter the accessibility of potential binding sites on DAF-16. These findings suggest that genetic modifications and exposure duration jointly modulate mixture toxicity, highlighting the need for cautious interpretation when using transgenic models in environmental risk assessment.